Bioactive surfaces made from surface-bound biomolecules may be used in a variety of bioassays, biosensors and other devices. For example, polymer-bound oligonucleotides find applications in hybridization-based diagnostics and in the discovery of new therapeutics based on molecular recognition. Prenatal diagnostics of genetic aberrations, identification of virus born diseases, detection of mutations of regulatory proteins controlling carcinogenesis, and novel hybridization-based identification techniques oriented to forensic or archaeology fields are some of the potential applications.
Bioactive surfaces may also play an essential role in areas other than medicine, pharmaceutics and biotechnology. Development of ultra-selective chemical sensors and absorbent surfaces are crucial for creating environmentally safe processes. Monitoring the quality of water is one of the major demands in this area. Biomolecular-based chemical sensors and filters for toxic chemicals and microorganisms (e.g., E. coli) will play a significant role in future technologies.
Proteins, and enzymes in particular, are one class of biomolecules commonly used to make bioactive surfaces. The advantages of using enzymes in bioassays and biosensors are related to their very high specificity (regio- and stereo-specificity) and versatility, mild reaction conditions (close to room temperatures and to pH neutral media), and to their high reaction rates. However, due to the poor recovery yields and reusability of free enzymes, much attention has been paid in the last few years to the development of efficient enzyme immobilization processes. Most biologically-active in vivo species, such as enzymes and antibodies, function in heterogeneous media. These environments are difficult to reproduce in vitro for industrial utilization. Immobilized enzyme systems are useful for experimental and theoretical research purposes for understanding the mechanisms of in vivo, bio-catalyzed reactions, and offer solutions for use in batch-type reactions, where there is poor adaptability to various technological designs and recovery of the enzymes is difficult.
The activity of enzymes (polypeptide molecules) are based on their complex three-dimensional structures containing sterically exposed, specific functionalities. The polypeptide chains are folded into one or several discrete units (domains), which represent the basic functional and three-dimensional structural entities. The cores of domains are composed of a combination of motifs which are combinations of secondary structure elements with a specific geometric arrangement. The molecular-structure-driven chain-folding mechanisms generate three-dimensional enzyme structures with protein molecules orienting their hydrophobic side chains toward the interior and exposing a hydrophilic surface. The —C(R)—CO—NH— based main chain is also organized into a secondary structure to neutralize its polar components through hydrogen bonds. These structural characteristics are extremely important and they make the enzyme molecules very sensitive to the morphological and functional characteristics of the potential immobilizing substrates. High surface-concentrations of enzyme-anchoring functionalities can result, for instance, in excessive enzyme-densities or multi-point connections which can “neutralize” the active sites or can alter the three-dimensional morphologies of the enzyme molecules through their mutual interaction and their interaction with the substrate surfaces. These are just a few of the factors which may be responsible for the significantly lower activities of immobilized-enzymes in comparison to the activities of free enzyme molecules. Rough substrate surface topographies or stereoregular surfaces (e.g., isotactic or syndiotactic polymers) might also influence, in a positive or negative way, the specific activities. Morphologically ordered surfaces might induce changes of the stereoregular shapes of protein molecules. It has also been found that enzymes can adopt more than one functional conformation other than its lowest potential energy state. E. S. Young, et al., Anal. Chem. Vol. 69, 1977, pp. 4242, et seq.
A number of approaches have been proposed for immobilizing bioactive molecules, such as enzymes on polymeric substrates. Most natural and synthetic polymeric substrates can easily be functionalized through polymer-analog reactions. Main chain and side-group homogeneous reactions are the most common approaches. The use of polymers as “carriers” or “supports” for chemical reagents, catalysts or substrates represents a relatively new, significant, and rapidly developing area. The polymer is in the form of an insoluble, inert substrate that may be a solvent-swollen, crosslinked gel, or a surface active solid. This approach eases the separation of reagents or catalysts (e.g., enzymes) from the reaction products, permitting the automation of the complex chemistry. However, the specific structure of the repeating units of the macromolecules often limit considerably the variety of polymer-analog reactions. These reactions are even more difficult to develop under heterogeneous environments. Natural and even some synthetic polymeric substrates can also undergo undesired chemical modifications, and sometimes biodegradation, during the polymer-supported organic reactions. Moreover, inert polymeric substrates (e.g., polyethylene, polypropylene, polyethylene terephthalate (PET), and polytetrafluoroethylene (PTFE)) and inorganic supports (e.g., glass, silica) cannot be functionalized efficiently by using conventional wet chemistry approaches.
The most widely used synthetic polymer surfaces are usually characterized by low surface energy values, and some of the thermoplastics, including polyethylene and polypropylene, for example, are essentially chemically inert. Modification of characteristics like adhesion, wettability, dyeability, and reactivity for such materials necessitates the creation of particular functionalities on the surfaces of such polymer substrates.
Cold plasma processing has shown promise for the functionalization of organic and inorganic substrates. See, e.g., D. T. Clark, et al., Polymer Surfaces (book), John Wiley & Sons, New York, 1978, pp. 185-210; F. Denes, et al., “Surface Modification of Polysaccharides Under Cold Plasma Conditions,” in Polysaccharides. Structural Diversity and Functional Versatility (book), Eds. Dumitriu, Marcel Dekker, Inc., New York, 1998; Plasma Surface Modification of Polymers: Relevance to Adhesion (book), Eds. M. Strobel, et al., VSP, Utrecht, The Netherlands, 1994; F. Denes, TRIP, Vol., No. 1, 1997, pp. 23, et seq. Numerous experiments performed in recent years in plasma laboratories under various internal and external plasma conditions and reactor geometries clearly indicate that inert and reactive-gas discharges are effective for the surface modification (functionalization) of even the most inert materials, such as polypropylene, Teflon®, silica, etc. The industrial applications of macromolecular plasma chemistry are rapidly developing. Large capacity reactors and continuous flow system plasma installations have been designed, developed and tested.
Several polymeric substrates have already successfully been functionalized with biomolecules using plasma techniques. For example, active horseradish peroxidase has been immobilized on acrylic acid and acrylamide radiation-grafted polymer surfaces. See H. Hongfei, et al., Radiat. Phys. Chem., Vol. 31, 1988, pp. 761 and A. A. Alencar, et al., Radiat. Phys. Chem., Vol. 55, 1999, p. 345. In these studies a cold plasma-technique was used for the surface functionalization. Immobilization of bioactive molecules onto synthetic and natural polymeric material surfaces often requires the presence of primary amine functionalities. Early attempts considered for the plasma-enhanced implantation of primary amine functionalities were ammonia discharge environments. However, due to the extensive fragmentation of NH3, other saturated, non-saturated and aromatic amines were also used as primary amine group precursors. Recently it has been shown that hydrazine-RF-plasmas are more adequate in comparison to ammonia discharges for the generation of surface primary amine functionalities on synthetic polymer surfaces. See Denes et al., J. Photopolym. Sci. Technol., Vol. 12, 1999, pp. 27 and Martinez et al., J. Biomater. Sci.: Polym. Ed., Vol. 11, 2000, pp.415.
Non-equilibrium plasma-mediated surface functionalization reactions have their shortcomings, however. Most of the precursor molecules of the desired surface functionalities (e.g., ammonia, hydrazine, saturated and non-saturated amines, etc.) undergo plasma-induced, intense fragmentation processes. As a consequence, undesirable functionalities will be implanted onto the substrate surfaces. These processes are also accompanied by the production of extremely reactive surface-free-radicals, and charged centers, which can induce further active-surface-mediated chemical reactions with the gas-phase plasma components through a variety of pathways. Plasma-generated free radicals (surface and stable, caged free radicals) can also initiate non-specific interactions with target molecules (e.g., biomolecules) under in situ or ex situ environments in the absence of plasma, which significantly diminishes the molecular recognition capabilities of the modified substrates.